RU2748289C1 - Canavan disease preimplantation genetic testing method - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to the field of medicine. A test system for the diagnosis of pathogenic variants in the ASPA gene associated with Canavan disease has been proposed.EFFECT: invention provides the creation of a test system for the diagnosis of pathogenic variants NC_000017.10: g.3392637G> T, NM_000049.2: c.634 + 1G> T, NC_000017.10: g.3402327C> A, NM_000049.2: c.857C> A, p.Ala286Asp in the ASPA gene with a dual detection system - direct and indirect.1 cl, 1 ex

Description

The invention relates to preimplantation genetic testing of monogenic diseases. There are currently over 350 million people worldwide with this rare disease (according to the RARE Project). The total number of such diseases, according to the estimates of the European Organization for Rare Diseases (EURORDIS), varies from 5 to 7 thousand. Moreover, about 80% of rare diseases have a genetic cause. The known genetic basis of the disease makes it possible to predict with high accuracy not only the health of an already born child, but also to assess the risk of having such a child when analyzing the genotypes of the parents, as well as to carry out genetic diagnostics at the earliest stages. Preimplantation genetic testing (PGT) for monogenic disease is becoming a powerful tool for the prevention of such diseases.

The present invention relates to a method for preimplantation genetic testing for Canavan disease. Canavan disease is an autosomal recessive disorder associated with impaired brain development. It belongs to the group of leukodystrophies. Canavan disease occurs with a frequency of 1: 6000 to 1: 14000 in Ashkenazi Jews and more rarely in other populations. Moreover, Canavan disease is the most common childhood degenerative disease. This disorder results in lethargy, lethargy, weak crying, lack of control over the head, hypotension, poor vision, vomiting, and seizures. The first symptoms appear around the age of three months. Macrocephaly becomes noticeable by 6 months, and by the same age, neurological disorders lead to a complete absence of motor development or significantly slow it down. Blindness develops by the age of 6-18 months. Fainting occurs in about 50% of patients. Death can occur during childhood, depending on the severity of the symptoms, but mild forms can also occur in adults. With an autosomal recessive type of inheritance of the disease, the probability of having a child with this disease in the family is 25%.

Canavan disease can be caused by pathogenic genetic variants in the gene ASPA (Aspartoacylase, Aspartoacylase), located on chromosome 17. This gene encodes the protein aspartic acid, a deficiency of which leads to the accumulation of N-acetyl-L-aspartate in the brain, causing dysfunction of oligodendrocytes and degradation in the phospholipid layer of axons.

Canavan disease OGT is performed for families with a confirmed molecular genetic nature of the disease. It is important to note that the substantiation of the pathogenicity and causation of genetic variants occurs before the OGT of a monogenic disease and is not included either in the goals and objectives of the OGT of a monogenic disease, or in the complex of measures for the OGT of a monogenic disease. Assessment of pathogenicity is carried out according to the international standard - according to the criteria described in 2015 by the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) [Richards S et al, 2015] during the search the molecular genetic cause of the disease. OGT is recommended for families with a high risk of having a child with a severe (incurable) hereditary disease with established pathogenic variants that cause this risk. OGT allows you to choose from all embryos obtained by IVF, embryos without pathogenic variants in the compound heterozygote and, therefore, without the risk of developing the disease.

The main problem in the genetic diagnosis of embryos is the small initial amount of biomaterial, since the biopsy specimen contains from one to three cells. In this case, in order to increase the efficiency and accuracy of the analysis, it is important both to completely exclude the possibility of contamination and to neutralize the possible effect of uneven and / or incomplete amplification, as well as degradation of the biomaterial. This requires the development of a test system with special characteristics. At the same time, the test system is being developed taking into account the possibility of using different types of biomaterial - total DNA isolated from different tissues, a product of Whole Genome Amplification (WGA), as well as single cells. The combination of versatility in relation to a biomaterial with a step-by-step amplification of target fragments makes it possible to analyze several pathogenic variants for one sample, including on single cells, as well as to reveal a situation of incomplete amplification, contamination or degradation of the sample. Another feature of OGT is the lack of information about the biological characteristics of the embryo: unlike an adult, an embryo can have any chromosomal abnormalities that complicate the task of assessing the status of an embryo for a specific genetic variant. Therefore, the test system for OGT of a monogenic disease should make it possible to identify such cases and assess their impact on the reliability of the diagnostic result.

The closest technical solution is the OGT of Canavan disease, proposed by Yaron and colleagues [Yaron Y. et al., 2005], using multiplex nested PCR in 2 rounds. An important advantage of our approach is the use of not only direct diagnosis, but also the use of indirect diagnostics by analyzing the inheritance of alleles of polymorphic markers linked to a pathogenic variant. This approach makes it possible to increase the reliability of the PGT-M result by increasing the likelihood of detecting a case of ADO (allele drop-out) - loss of an allele due to insufficient amplification - with a direct test for the presence of a pathogenic variant in an embryo. Also, the use of indirect diagnostics is the basis for the versatility of the test system we offer, since it allows PGT-M for any pathogenic variant of the ASPA gene.

The presented method of PGT of Canavan disease solves the problem of developing a more accurate method of preimplantation genetic testing of this monogenic disease without the use of expensive devices and reagents, which could be used on various types of biomaterial: DNA isolated from different tissues, the product of whole genome amplification (WGA), single cells.

The technical result was the creation of a test system for the diagnosis of pathogenic variants (NC_000017.10: g.3392637G> T (NM_000049.2: c.634 + 1G> T), NC_000017.10: g.3402327C> A (NM_000049.2: c .857C> A, p.Ala286Asp) in the ASPA gene with a dual detection system - direct and indirect.This system is necessary when working with a small amount of biomaterial, since unstable amplification can lead to loss of information or reduced accuracy of the analysis.Direct diagnosis implies analysis directly presence-absence of a pathogenic variant.In this case, for genetic variants NC_000017.10: g.3392637G> T (NM_000049.2: c.634 + 1G> T), NC_000017.10: g.3402327C> A (NM_000049.2: c .857C> A, p.Ala286Asp) single nucleotide polymorphism (SNP), restriction endonucleases were selected, which allow the detection of a pathogenic variant by the method of RFLP-RFLP (restriction fragment length polymorphism) based on the sequence difference in the restriction site different countries llelei. Indirect diagnosis consists in the analysis of the inheritance of molecular genetic markers linked to the mutation. For this, polymorphic loci (markers) called STR (short tandem repeat) with heterozygosity of at least 0.70 were selected in each direction at a distance of no more than 3 Mb (which corresponds to 3% crossing over on average) from the ASPA gene. to ensure maximum information content of indirect diagnostics. For each of these loci, primers were selected for amplification according to the type of nested or semi-nested PNR in 2 rounds, which makes it possible to increase the accuracy and efficiency of amplification. The test system included 14 STR loci for the ASPA gene: D17S1.7, D17S1.8, D17S654, D17S1.9, D17S2.1, D17S2.3, D17S2.8, D17S1828, D17S4.4, D17S4.6, D17S559 , D17S4.7, D17S5.1, D17S5.2. A list of primer sequences with an indication of the marker or pathogenic variant for which these primers were used (external primers are designated as Fout (forward primer) and Rout (reverse primer), and internal - as Fin (forward primer) and Rin (reverse primer)) are presented in SEQ ID NO 1-50.

A combination of direct and indirect diagnostics is carried out to increase the reliability of the result, taking into account the complications typical for diagnostics under conditions of OGT. For nested or semi-nested PCR amplification, primers SEQ ID NOs 1-50 were used. Direct diagnosis involves the analysis of the presence of the most pathogenic variant using the PCR-RFLP method. Indirect diagnostics makes it possible to analyze the inheritance of molecular genetic polymorphic markers of the STR type linked to a pathogenic variant. It is important to note that a number of special requirements were observed in the selection of primers: the length of the product with external primers for the first round of PCR should not exceed 500 bp. (for production from fragments obtained by whole-genome amplification), the length of the product from internal primers for the second round of PCR is from 120 to 350 bp, high specificity of external primers, the annealing temperature of primers in each pair does not differ by more than 1 ° С ...

Preparatory stage of PGT

At the preparatory stage, the test system is being worked out: the selection of amplification conditions that are optimal for the operation of the primers, the analysis of the efficiency and specificity of PCR amplification in both rounds, the assessment of the versatility of the test system for biological samples of various types (DNA, WGA product, single cells). When testing the test system, stock dilutions of primers with a concentration of 100 mM were prepared, and working dilutions of primer combinations (a combination of primer pairs for rounds 1 and 2 of PCR) with a concentration of 10 mM of each primer in solution. Since various types of matrices can be used in the diagnosis of clinical material, two biopsies of single cells were used in the development of the test system in a special lysis buffer (1 × PCR Buffer, 0.1% Tween-20, 0.1% Triton X-100, 1 μg Proteinase K), two samples of the products of genome-wide amplification of embryo biopsy (WGA), as well as total DNA of family members isolated from blood, to compile a pedigree and identify the linkage of a pathogenic variant with alleles of polymorphic markers.

In nested and semi-nested PCR, amplification is carried out in two stages. At the first stage, multiplex PCR is carried out with all external primers for all loci included in the test system to enrich the sample with all target fragments. At the second stage, each fragment is individually amplified with internal primers.

Semi-nested PCR

For the first stage, external highly specific primers were selected to amplify fragments from 300 to 500 bp. For the second stage, primers were selected to amplify fragments no more than 350 base pairs in length, and labels were introduced for detection by fragment analysis. The PCR mixture for the first round of amplification contained 1 × PCR Buffer with Mg2 + (Evrogen, Russia), 0.1 mM of each deoxynucleotide, 0.15 μM of each primer, 2.5 U / μl of HsTaq DNA-polymerase (Evrogen, Russia), 6% of DMSO and 1 μl total DNA or 2.5 μl WGA or 5 μl lysis buffer with sample as template. The first stage of amplification was carried out according to the following protocol: the stage of denaturation at 94 ° C for 2 minutes, 30 cycles with a decrease in the primer annealing temperature from 62 to 45 ° C in each, the stage of completing all templates 72 ° C for 10 minutes. Further, the products of the 1st stage were distributed into individual tubes with one pair of primers at a certain locus.

The PCR mixture for the second stage consisted of 1 × PCR buffer with Mg2 + (Evrogen, Russia), 0.5 × RediLoad ™ loading buffer (Thermo Fisher Scientific, USA), 0.2 mM of each deoxynucleotide, 0.2 μM of each primer, 1U / μl of HsTaq DNA polymerase (Evrogen, Russia), 6% dimethyl sulfoxide (DMSO) and 1 μl of the PCR product of the first amplification stage as a template. The second stage of amplification was carried out according to the following protocol: stage of denaturation at 95 ° С for 2 minutes, 35 cycles: denaturation at 95 ° С for 30 seconds, annealing of primers - 57 ° С for 30 seconds, template synthesis - 72 ° С for 1 minute, stage of completion of all matrices 72 ° C 5 minutes. Evaluation of the efficiency and specificity of amplification was carried out using electrophoresis in 2% agarose gel. The result of electrophoresis in agarose gel makes it possible to determine the required dilution of the amplification products for application to the fragment analysis (amplification products of the DNA of family members).

Fragment analysis of amplification products was performed using capillary electrophoresis on a 3130 × l Genetic Analyzer (Applied Biosystems, USA). Based on the results of the fragmentary analysis, a pedigree is compiled and informative polymorphic STR-loci for each family are noted, which will be further used in clinical diagnostics. Loci are divided into uninformative (the carrier of the pathogenic variant is homozygous for this locus), semi-informative (on some and parental chromosomes, alleles for this marker coincide), informative (on all chromosomes of parents, alleles of this marker are different, which makes it possible to distinguish each of them when analyzing the genotype of the embryo ).

Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP)

Restriction fragment length polymorphism (RFLP) is a method for studying genomic DNA by specifically cleaving DNA with restriction endonucleases and further analyzing the size of the resulting fragments (restriction) by gel electrophoresis. When using this method, fragments of different lengths are observed depending on differences in the nucleotide sequence at the restriction site, which makes it possible to detect single nucleotide variants if they are located at the restriction site. More accurate detection of the pathogenic variant can be provided by sequencing according to the Sanger method, however, under the conditions of OGT, the PCR-RFLP method turns out to be more effective due to the reduced probability of allele drop out (ADO) and, as a consequence, an erroneous result in assessing the status of the embryo by pathogenic option.

For the detection of pathogenic variants NC_000017.10: g.3392637G> T (NM_000049.2: c.634 + 1G> T), NC_000017.10: g.3402327C> A (NM_000049.2: c.857C> A, p.Ala286Asp ) a test system based on PCR-RFLP was developed. The amplification step is described in detail in the previous section. The following primers were used:

• For option NC_000017.10: g.3392637G> T (NM_000049.2: c.634 + lG> T)

Внешние праймеры:

External primers:

5'-TGTTCTGCTTCACGTTTTGC-3 '(Fout),

5'-TATCCTGGCCATTGGGGAAT-3 '(Rout),

Внутренние праймеры:

Internal primers:

5'-ТСAAGGGGTTCTGAGAGCTG-3 '(fin),

5'-TTTGATAACGTTACCTTCATTATTAGTTA-3 '(rin_mm_mut)

• For option NC_000017.10: g.3402327C> A (NM_000049.2: c.857C> A, p.Ala286Asp)

Внешние праймеры:

External primers:

5'-TCCTGGGGATCCCATGTTTT-3 '(Fout),

5'-ATGTGCTTAGATGCCTACCG-3 '(Rout),

Внутренние праймеры:

Internal primers:

5'-GATGGGAAGACGATCCCACT-3 '(fin),

5'-AGATGCCTACCGAATAAGGC-3 '(rin).

Further, the products of amplification from internal primers were used in the restriction reaction to detect the pathogenic variant. Endonuclease Tru1I cuts only the wild-type allele, HincII endonuclease cuts only the mutant allele of the NC_000017.10 variant: g.3392637G> T (NM_000049.2: c.634 + 1G> T). The BsuRI endonuclease cuts only the wild-type allele; the CseI endonuclease cuts only the mutant allele of the NC_000017.10 variant: g.3402327C> A (NM_000049.2: c.857C> A, p.Ala286Asp). Detection was carried out by electrophoresis in 12% polyacrylamide gel.

Example

Patient A

Family A, in which each of the parents was a carrier of one of the pathogenic variants NC_000017.10: g.3392637G> T (NM_000049.2: c.634 + 1G> T) and NC_000017.10: g.3402327C> A (NM_000049.2: c.857C> A, p.Ala286Asp) in the ASPA gene. The couple was recommended to carry out OGT of the disease Canavan disease within the framework of IVF for the selection of embryos that did not inherit the disease.

Family haplotyping

At the first stage, a biomaterial (peripheral blood) of family members was obtained to detect the pathogenic variant and identify linkage groups of alleles of polymorphic markers. 14 STR loci were analyzed. Of these, 10 turned out to be informative for the mother and / or father. Thus, embryo samples were tested for informative markers only. The results obtained for informative markers (the alleles indicated on one side of the “/” symbol for different markers are located on the same chromosome, that is, they form a linkage group): the patient's partner (carrier of the pathogenic variant ASPA c. 857C> A (p.Ala286Asp) ): D17S1.8 - 170/175, D17S654 - 284/277, D17S1.9 - 160/175, D17S2.1 - 189/193, D17S2.3 - 216/225, ASPA - p.A286D / N, D17S1828 - 121/123, D17S4.6 - 359/355, D17S559 - 221/221, D17S4.7 - 185/185, D17S5.1-277/273, patient (carrier of the pathogenic variant c.634 + 1G> T): D17S1. 8> 170/173, D17S654 - 284/277, D17S1.9 - 182/175, D17S2.1 - 191/189, D17S2.3 - 116/216, ASPA - N / c. 634 + 1G> T, D17S1828 - 119/127, D17S4.6 - 359/363, D17S559 - 231/229, D17S4.7 - 185/179, D17S5.1 - 270/277, a child without disease (carrier of the pathogenic variant cp.A286D): D17S1.8 - 171/171, D17S654 - 284/284, D17S1.9 - 161/182, D17S2.1 - 189/191, D17S2.3 - 216/216, ASPA - p.A286D / N, D17S1828 - 121/119, D17S4. 6 - 359/359, D17S559 -221/231, D17S4.7 - 185/185, D17S5.1 - 277/270, child without disease (carrier of pathogenic variant c.634 + 1G> T): D17S1.8 - 175/173, D17S654 - 277/277, D17S1.9 - 176/176, D17S2.1 - 193/189, D17S2.3 - 225/216, ASPA - N / c. 634 + 1G> T, D17S1828 - 123/127, D17S4.6 - 355/363, D17S559 - 221/229, D17S4.7 - 185 / 179, D17S5.1 - 273/277.

Preimplantation genetic testing

In the IVF cycle, 3 embryos were obtained, a biopsy was performed on the 5th day of development (at the IVF clinic), a biopsy sample in WGA buffer (1 × PBS (Invitrogen, USA), 1% polyvinylpyrrolidone (PVP) (Fertipro, Belgium)) was sent to the laboratory Genetico. To control contamination at different stages of working with a sample, a control system has been developed in the laboratory: control of contamination of the biopsy buffer, control of contamination during transportation (one tube with the buffer cannot be opened by the embryologist), control of the contamination of each sample (sample of the medium from the last washout drop of biopsy material). All these controls, together with the samples, undergo a genome-wide amplification stage, after which the slightest amount of DNA contaminating the controls will be noticeable. Whole genome amplification was performed using a commercial SurePlex kit (Illumina, USA).

The whole genome amplification product, as well as the DNA of all family members, were amplified at the first stage in a multiplex PCR with primers for detecting the pathogenic variant and primers for polymorphic markers informative for family A in accordance with the protocol for the test system developed during the preparatory stage. At the second stage, amplification was performed for each marker separately in accordance with the developed protocol for the test system. In this way, the linkage groups inherited by each embryo were established. Results obtained: embryo1: D17S1.8 - 170/173, D17S654 - 277D17S1.9 - 175, D17S2.1 - 193 / 189D17S2.3 - ADO / 216, ASPA - N / c.634 + 1G> TD17S1828 - 123/127 , D17S4.6 - 355 / 363D17S559 - 221/229, D17S4.7 - 185 / 179D17S5.1 - 273/277; embryo2: D17S1.8 - 170/173, D17S654 - 284/277, D17S1.9 - 160/175, D17S2.1 - 189, D17S2.3 - 216, ASPA -p.A286D / c.634 + 1G> T, D17S1828 - 121/127, D17S4.6 - 359/363, D17S559 - 221/229, D17S4.7 - 185/179, D17S5.1 - 277; embryo3: D17S1.8 - 175/173, D17S654 - 277, D17S1.9 - 175, D17S2.1 - 193/189, D17S2.3 - 225/216, ASPA - N / c.634 + 1G> T, D17S1828 - 123/127, D17S4.6 - 355/363, D17S559 - 221/229, D17S4.7 - 185/179, D17S5.1 - 273/277.

According to the results of direct and indirect diagnostics, 2 embryos did not inherit the disease, 1 embryo had a haplotype corresponding to the inherited disease. With parental consent, preimplantation genetic screening for chromosomal abnormalities was performed for embryos that did not inherit the disease. Based on the results of all the analyzes carried out, 1 embryo was recommended for transfer.

Primer Sequence Listing

external primers are designated as Fout (forward primer) and Rout (reverse primer), and internal primers as Fin (forward primer) and Rin (reverse primer

• D17S1.7
SEQ ID NO 1: 5'-TCAGGTGGGGATAAAGGTC-3 '(Fout);
SEQ ID NO 2: 5'-TCCCACTAACCTCCCTCC-3 '(Rout)
SEQ ID NO 3: 5 '- * CCTGATCACGGACACATCAC-3'(Fin);
SEQ ID NO: 4 5'-TCTTGTGTTCTCACCCCTGA-3 '(Rin)

• D17S1.8
SEQ ID NO 5: 5'-AGGCTGTCTGTTTCATGAGC-3 '(Fout);
SEQ ID NO 6: 5'-TCTGGTCAGAGATATTGTGGG-3 '(Rout)
SEQ ID NO 7: 5 '- * CCAAGGGTCCTTCTAGCTCT-3' (Fin)

• D17S654
SEQ ID NO 8 5'-ACCTTCCTCCTCTGGATCAC-3 '(Fout);
SEQ ID NO 9 5'-GGTTGGCAAGACCACTGTTA-3 '(Rout);
SEQ ID NO 10 5 '- * GCCTACCCACTTTGTGAGTTT-3' (Rin)

• D17S1.9
SEQ ID NO 11 5'-TCTGGGTATTCGGAGACACT-3 '(Fout);
SEQ ID NO 12 5'-TGGTGCTCTTTAACCTGCTC-3 '(Rout);
SEQ ID NO 13 5 '- * TACACCCATCTACCACTGCTT-3' (Rin)

• D17S2.1
SEQ ID NO 14 5'-CTGCAGTAACACCTGGAACTC-3 '(Fout);
SEQ ID NO 15 5'-TCTGGACCCTGAAAACTCCT-3 '(Rout)
SEQ ID NO 16 5'-CTGCAGGGAATGTCTCCAT-3 '(Fin);
SEQ ID NO 17 5 '- * AGGATACCGGGAAGGTTTG-3' (Rin)

• D17S2.3
SEQ ID NO 18 5'-ACTGGGGGACGTCTTCTAATA-3 '(Fout);
SEQ ID NO 19 5'-TGCAAAGCGTTAGGCTCTC-3 '(Rout);
SEQ ID NO 20 5 '- * ACAAGTTCAGGTTCAGACAGGT-3' (Rin)

• D17S2.8
SEQ ID NO 21 5'-AGATGGAAAGTGGGGAGTG-3 '(Fout);
SEQ ID NO 225'-CGGATTCATTCTCCCTTGA-3 '(Rout)
SEQ ID NO 23 5 '- * GGACAGCTTTTTGACCAGG-3' (Fin)

• D17S1828
SEQ ID NO 24 5'-TCCTCTCCAGGCTGTGG-3 '(Fout);
SEQ ID NO 25 5'-TAGAGAAGAGGTGGGTGGG-3 '(Rout)
SEQ ID NO 26 5'-ACTCTTTTTGGAGATTCAGGG-3 '(Fin);
SEQ ID NO 27 5 '- * CACAGGTGCACTCACAGATT-3' (Rin)

• D17S4.4
SEQ ID NO 28 5'-TTGTCCATCCATCCAGCA-3 '(Fout);
SEQ ID NO 29 5'-GTCTGAGGGAACACAAGCAG-3 '(Rout)
SEQ ID NO 30 5'-CAGCCTAATGGCTGAGACAG-3 '(Fin);
SEQ ID NO 31 5 '- * AAAGGACAGAAGGAGTGGGA-3' (Rin)

• D17S4.6
SEQ ID NO 32 5'-CCGGGCTATGGCCTATT-3 '(Fout);
SEQ ID NO 33 5'-TCAGACTAGTGGACCTTTAGGCT-3 '(Rout)
SEQ ID NO 34 5'-GCTCTCTAGCTGGCTGGTT-3 '(Fin);
SEQ ID NO 35 5 '- * CTCCTTTATCTCTGCACACCTC-3' (Rin)

• D17S559
SEQ ID NO 36 5'-GAGATTTTGTCCAGTGGCTAGA-3 '(Fout);
SEQ ID NO 37 5'-TGACATGATTTACGTCTCTCTCAA-3 '(Rout)
SEQ ID NO 38 5 '- * GTGCTACGTGTGGGTAGGAT-3'(Fin);
SEQ ID NO 39 5'-TTGTTATATGTGCCCACGG-3 '(Rin)

• D17S4.7
SEQ ID NO 40: 5'-CCCCACCCCTGTCTTAAT-3 '(Fout);
SEQ ID NO 41: 5'-ATATGGCTTCAGTGGGACC-3 '(Rout)
SEQ ID NO 42: 5'-GACACAGGGAGCTTTGTTCA-3 '(Fin);
SEQ ID NO: 43 5 '- * GGTTCCTTGAGCCTGATGAC-3' (Rin)

• D17S5.1
SEQ ID NO 44: 5'-GGAGTACACAATGGCTGCA-3 '(Fout);
SEQ ID NO 45: 5'-AACCTCCTGAGTAGCTGTGACTA-3 '(Rout)
SEQ ID NO 46: 5 '- * TGAGTACAGGACTACTGAGGGC-3' (Fin)

• D17S5.2
SEQ ID NO 47 5'-AATGGCCGTATGTTCTTTATGTA-3 '(Fout);
SEQ ID NO 48 5'-CACTCTCAATCTGGGCCG-3 '(Rout)
SEQ ID NO 49 5 '- * ACGTGCCAATTCAACCAT-3'(Fin);
SEQ ID NO 50 5'-TGGTAGCTAAAGGGCGAAG-3 '(Rin)

Claims (1)

Test system for diagnosing pathogenic variants NC_000017.10: g.3392637G> T, NM_000049.2: c.634 + 1G> T, NC_000017.10: g.3402327C> A, NM_000049.2: c.857C> A, p .Ala286Asp in the ASPA gene associated with Canavan disease, with a dual detection system - direct and indirect, including a PCR mixture for the first round of amplification, containing 1 × PCR buffer with Mg2 +, 0.1 mM of each deoxynucleotide, 0.15 μM of each primer , 2.5 U / μl HsTaq DNA polymerase, 6% dimethyl sulfoxide (DMSO) and 1 μl total DNA or 2.5 μl WGA or 5 μl lysis buffer with sample as template, PCR mixture of the second round of amplification containing 1 × PCR buffer with Mg2 +, 0.5 × RediLoad loading buffer, 0.2 mM of each deoxynucleotide, 0.2 μM of each primer, 1 U / μl of HsTaq DNA polymerase, 6% DMSO and 1 μl of the PCR product of the first amplification step as a template, wherein the primers for the loci are SEQ ID NOs 1-50.